Material and methods
In early October 2019, mortality of free-living red-legged partridges
was detected in a hunting estate in Cádiz (Southern Spain). Partridges
in fair to poor body condition were found dead, or with severe central
nervous signs, similar to those observed in the
BAGV-related mortality in the same
geographic area in 2010. The birds showed weakness, ruffled feathers,
apparent blindness and neurological signs such as ataxia that led to
loss of flight coordination or complete inability to fly.
We carried out full, detailed post-mortem examination on four individual
birds that had died after showing neurological signs or were found dead
and collected vascular growing feathers, heart, brain, liver, spleen and
kidney for pathogen detection in addition to a complete set of tissues
(heart, brain, spleen, liver, kidney, lung, skeletal muscle, skin, cecal
tonsils, adrenal glands, gonads and pancreas) for histopathology and
immunohistochemistry. No ethical approval was required as this study was
based on samples from deceased animals.
Whole DNA and RNA were manually extracted from pulp of growing feathers,
heart, brain, liver, spleen and kidney using TRI Reagent® (Merck Life
Science, Madrid, Spain) according to manufacturer’s instructions. We
tested for the presence of haemosporidian parasites using a nested PCR
with posterior sequencing of the 524 bp PCR product (Waldenström et al.,
2004). Sample RNA was analyzed using a generic flavivirus SYBR Green
(BioRad, Madrid, Spain) real-time reverse transcription PCR as described
previously (Moureau et al., 2007) and the new obtained sequences were
deposited in GenBank (acc. n. OK424741, OK424742). We used ClustalW to
align the sequences of the NS5 gene fragment obtained in this study
(highlighted in bold in Figure 2) and reference sequences from GenBank
including different variants of Bagaza virus, from different
localizations (partial segment, 222 bp). A total of 30 sequences were
used for this analysis, and sequence of West Nile virus was used as an
outgroup. A phylogenetic tree was generated using MEGA version 10
(http://www.megasoftware.net)
(Kumar et al., 2018) with the Maximum Likelihood method with Kimura
2-parameter distances (Kimura et al., 1980), this model was selected
based on lowest values of Corrected Akaike Information Criterion (cAIC)
and Bayesian Information Criterion (BIC). Reliability of internal
branches was assessed using the bootstrapping method (1000 replicates).
In the case of avian malaria, phanerozoites observed in the
hematoxilin-eosin-stained tissues were confirmed to be Plasmodiumspp. by immunohistochemistry (IHC) with the
avidin-biotin-peroxidase complex (ABC) method (anti-Plasmodiumspp. polyclonal antibody; LSBio, Lifespan Biosciences, Seattle,
Washington, USA). This method was also used to detect BAGV in different
tissues, using a polyclonal anti-WNV antibody and following the protocol
described by Gamino et al. (2013).